Lenalidomide, a derivative of thalidomide, is a transformative therapy for a subset of patients with myelodysplastic syndrome, and has demonstrated promise in early clinical trials in acute myeloid leukemia (AML), but the mechanism of lenalidomide activity in myeloid malignancies is not known. We have identified and validated an E3 ubiquitin ligase, CRL4-CRBN, as a direct target of lenalidomide, consistent with previous reports that this complex is targeted by thalidomide. We hypothesize that the pleitropic effects of lenalidomide, including its therapeutic efficacy in myeloid malignancies, is due to altered ubiquitination of targets ofthe CRL4-CRBN ubiqutin ligase. In Aim 1, we will use a recentiy developed proteomic approach to define the proteins that are differentially ubiquitinated in AML cells in response to lenalidomide, and we will validate that these proteins are direct targets of the CRL4-CRBN ubiquifin ligase using genetic tools and biochemical assays. In Aim 2, we will use similar approaches to define the molecular basis of the immunomodulatory properties of lenalidomide that lead to alterations in the bone marrow microenvironment. These effects may be critical for the therapeutic efficacy of lenalidomide. In Aim 3, we will investigate how altered ubiquitination of specific proteins may sensitize cells to additional therapies. Significant responses to lenalidomide as a single agent have been reported in AML, but only a subset of pafients respond, and complete remissions are of short duration. We will therefore seek to improve the therapeutic potential of lenalidomide by examing combinations with addifional therapies in collaboration with each of the other projects in this POl. In addition, we will examine ubiquitinated proteins and genetic abnormalities in patients treated with lenalidomide plus induction chemotherapy in a clinical trial proposed in Project 5. These studies will elucidate a novel mechanism for a cancer therapy, the direct targeting of a specific ubiquitin ligase with both cell autonomous and cell non-autonomous effects. In addition, we will identify novel combinations of lenalidomide with additional agents to develop more efficacious treatments for AML.